PROJECT SUMMARY: The interaction between tumor cells and macrophages has been shown to be crucial in promoting tumor invasion and metastasis. These two cell types are engaged in a paracrine interaction in which tumor- associated macrophages secrete epidermal growth factor (EGF) to activate tumor cells. In turn, tumor cells secrete colony-stimulating factor-1 (CSF-1) to which macrophages respond. While soluble factors are well known to transmit signals between different cells, it is unclear how these soluble mediators can lead to the co- migration of macrophages and tumor cells away from the high concentration of these factors present in the primary tumor. Recent studies done in our lab have revealed a novel mechanism of intercellular communication between macrophages and tumor cells that can transmit signals over long distances through membranous actin-based tunneling nanotubes (TNTs).These thin actin containing structures form rapidly but have long lifetimes and can be up to several cell diameters in length. Our preliminary data demonstrates that the formation of TNTs in macrophages is dependent on actin polymerization, through activation of the RhoGTPases Cdc42 and Rac1 and their downstream effectors WASP and WAVE. Using FRET-based biosensors, we have observed differential spatiotemporal activation of Cdc42 and Rac1 indicating that they play different roles during the formation of TNTs in macrophages. Interestingly, our preliminary data shows that heterotypic TNTs form between macrophages and tumor cells in co-culture. This novel interaction between macrophages and tumor cells via TNTs and can induce changes in tumor cell morphology consistent with a more invasive phenotype. The proposed work to be completed during the F99 phase of this fellowship will be focused on the regulation of heterotypic TNT formation and dynamics between macrophages and tumor cells, as well as the role of TNTs in mediating macrophage-dependent tumor cell migration and invasion. We will determine the structure and mechanism by which TNTs are formed between macrophages and tumor cells. We will also determine the spatiotemporal activation of RhoGTPases during the formation of heterotypic TNTs as well as the dependence of TNT formation on the paracrine loop which we know is required for mutual cell invasion. Importantly, we will analyze the effects of macrophage TNTs on tumor cell function including EGFR activation, RhoA activation, invadopodia formation, directional migration and invasion. In addition to 2D and 3D assays, we will employ a newly developed 1D assay that mimics the co-migration of tumor cells and macrophages along fibers as seen in vivo to determine the role of TNTs in the promotion of long distance paracrine invasion. Overall, the results of this study will increase our understanding of TNT formation and regulation in different cell types and determine whether TNTs are required to the persistent long range migration of macrophages and tumor cells allowing for tumor progression to metastasis. Aim 3 of this proposal will focus on the transition into the K00 phase of the fellowship describing my postdoctoral research direction following the completion of the F99 phase.